Lysyl hydroxylase 2 mediated collagen post-translational modifications and functional outcomes

Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.

Other modifying enzymes and associated proteins. We then analyzed the protein levels of LH1 and LH3 in KO clones by Western blot analysis (Fig. 2, Supplementary Fig. S3). The results showed that both LH1 and LH3 were comparable to controls (p > 0.05) though the former tended to be slightly lower in KO clones (Fig. 2). The collagen galactosyl transferase, GLT25D1, was significantly lower in the KO clones when compared to controls (Fig. 2). The reason for this is unclear, but the reduced level of GLT25D1 in KO could be partially compensated by unknown mechanisms since the total levels of G-+ GG-Hyl in KO collagen were only slightly lower (< 10%) than those of controls at all glycosylation sites analyzed (see below). The LH2-specific chaperone, FK506-binding protein 65 (FKBP65) 10 , and an additional potential binding partner, cyclophilin B (CypB) 33 , showed slightly but significantly lower (~ 70% of controls) or similar level (~ 90%), respectively, in KO clones when compared to controls (Fig. 2). Other LH2-associated proteins, heat shock protein 47 (Hsp47) and immunoglobulin heavy-chain-binding protein (Bip) 34 , were also significantly lower in KO than controls (Fig. 2).
All these measurements were conducted using cells cultured with the standard medium, thus, further analyses using those with the differentiation and mineralization media (see below) are necessary to obtain more comprehensive information.
Collagen type. We first examined collagen types by mass spectrometric analysis 35 . The data revealed that type I collagen is by far the predominant collagen type with a small amount of type III in all of the culture samples, which is consistent with our previous report 36 . The percentages of type I calculated by I/(I + III) × 100 were all > 96% and the difference between MC and KOs was within ~ 2% range ( www.nature.com/scientificreports/

Lys hydroxylation determined by high performance liquid chromatography (HPLC). In KO
clones, levels of Lys hydroxylation in collagen were slightly but significantly decreased compared with those from MC and EV (Table 2). We then analyzed Lys modifications at specific molecular loci in type I collagen (see below).

Lys modifications at specific molecular loci in type I collagen. Relative abundance of unmodified
Lys and its modified forms (Hyl, G-Hyl and GG-Hyl) at specific molecular sites of type I collagen are summarized in Table 3. Based on these values, an extent of Lys hydroxylation (%) was calculated as [Hyl/(Lys + Hyl) × 100] where Hyl is a sum of non-glycosylated, G-and GG-Hyl.
Lys hydroxylation in the telopeptides. None of the telopeptidyl Hyl is glycosylated (Table 3). Lys hydroxylation in the telopeptides of type I collagen, i.e. N-telo (α1 Lys-9 N and α2 Lys-5 N ) and C-telo (α1 Lys-16 C ) (note: α2 C-telo lacks Lys), is shown in Fig. 3a. The values of MC and EV were essentially identical with no statistical difference, i.e. ~ 55.4% at α1 Lys-9 N , ~ 22.7% at α2 Lys-5 N and ~ 56.8% at α1 Lys-16 C (Fig. 3a). In the KO type I collagen, however, none of the Lys residues was hydroxylated at any of these sites (Table 3, Fig. 3a). These results unequivocally demonstrate that LH2 is responsible for Lys hydroxylation in all telopeptides of type I collagen and that other LHs cannot compensate for this function.
Lys modifications in the helical domain. We then analyzed Lys modifications in the helical domain of type I collagen by using tryptic digests of collagen 8,33,37 (Table 3, Fig. 3b). In the helical domain, modified Lys residues were identified at 11 sites. The values in Fig. 3 represent percentages calculated as [Hyl/(Hyl + Lys) × 100] where Hyl includes glycosylated (G-and GG-) and non-glycosylated forms (Table 3). First, we examined the helical cross-linking sites, i.e. α1 Lys-87, α1 Lys-930, α2 Lys-87 and α2 Lys-933. At α1 Lys-87, a highly hydroxylated and the most heavily glycosylated site of type I collagen 16,17 , ~ 98% of Lys was hydroxylated in controls, MC and EV. In KO collagen, it was also almost all hydroxylated, showing only 2-4% less hydroxylated than controls (Table 3). For α1 Lys-930, using the collagenase-pepsin digest 37 , we analyzed Lys hydroxylation in the peptide containing α1 Lys-918/930 (GDKGETGEQGDRGIKGHR). In controls, these Lys residues were at least 87-89% hydroxylated (Hyl + Hyl), and those in KO (Fig. 3b). These data indicate that the contribution of LH2 towards helical Lys hydroxylation is low, especially at the cross-linking sites, and site-specific at non-crosslinking sites.   (Table 4). When calculated as percentages of non-glycosylated-Hyl and glycosylated (G-and GG-) forms in total Hyl, the relative abundance of glycosylated Hyl at α1 Lys-87, the Values represent mean ± S.D. (n = 3) from three independent experiments. Statistical differences were determined by the method described above (Fig. 1 legend). *p < 0.05, **p < 0.01, and ***p < 0.001 between MC and KO; # p < 0.05, ## p < 0.01, and ### p < 0.001 between EV and KO, respectively. Original blots are presented in Supplementary Fig. S3. LH lysyl hydroxylase, GLT25D1 glycosyltransferase 25 domain containing 1, CypB cyclophilin B, FKBP65 FK506-binding protein 65, Hsp47 heat shock protein 47, Bip immunoglobulin heavychain-binding protein, Ab antibody, MC MC3T3-E1, EV empty vector, KO knock-out. www.nature.com/scientificreports/ major glycosylation site, was slightly but significantly lower (2-10%) and non-glycosylated-Hyl significantly higher in KO collagen compared to those of controls (Table 4). At all other sites, i.e. α1 Lys-99, α1 Lys-174, α1 Lys-564, α2 Lys-174, and α2 Lys-219, the same phenomena, i.e. a lower level of glycosylation of Hyl in KO collagen, were observed (Table 4) with the exception of KO-3 exhibiting similar levels of non-glycosylated and glycosylated Hyl to controls at some sites (p > 0.05, respectively). These data indicate that LH2 deficiency may cause diminished glycosylation at several sites. Interestingly, when a percentage of two glycosylation forms (G-+ GG-= 100%) was calculated, GG-form was lower and G-form higher at most sites in KO collagen when compared to controls ( Table 5). These data suggest that LH2 deficiency causes a relative decrease of galactosylhydroxylysineglucosyl transferase (GGT) activity leading to relative increase in the G-Hyl form, which is consistent with our recent report 6 . Collagen cross-link analysis. Control groups (MC and EV) showed essentially identical cross-link patterns ( Fig. 4) with no statistical difference in any of the cross-links. The amounts of cross-links of control and KO collagens are summarized in Table 7. In control groups, the major cross-link was DHLNL (Hyl ald × Hyl) representing ~ 67% of the total cross-links. The rest includes HLNL (Hyl ald × Lys or Lys ald × Hyl), Pyr (Hyl ald × Hyl ald × Hyl) and HHMD (Lys ald × Lys ald × His × Hyl). In KO collagen, none of the Hyl ald -derived cross-links (DHLNL, Pyr) were detected while Lys ald -derived cross-links, HLNL and HHMD, were both significantly increased by ~ 44 and ~ 400%, respectively. Though HLNL can be derived from Hyl ald or Lys ald , since Lys at the helical crosslinking sites are almost fully hydroxylated and telopeptidyl Lys is not hydroxylated in KO collagen (Table 3), it should be derived from Lys ald × Hyl in KO. In contrast to the striking difference in the type of cross-links, the difference in the total number of aldehydes involved in cross-linking is small (0.1-0.2 mol/mole of collagen) between control and KO collagens. This indicates that LOX/LOXL activities are not significantly affected in KO clones.

Pro 3-hydroxylation.
Collagen solubility, fibrillogenesis and matrix mineralization. We then evaluated the biochemical, morphological, and functional outcomes of LH2KO. First, we found that LH2KO resulted in a marked increase in collagen solubility (  Fig. 5. The fibrils in KO clones were generally circular in shape and overall similar to those of MC and EV. However, the collagen fibril diameters in all KO clones were smaller than those of MC and EV (Fig. 5), indicating defective lateral growth of fibrils in KO collagen. Lastly, we assessed the effects of LH2KO on in vitro mineralization. The controls (MC and EV) and KO clones (1-3) were cultured for 28 days and subjected to mineralization assay using Alizarin red S staining (Fig. 6). In the controls (MC and EV), mineralized nodules were well formed at this point, however, no nodules were observed in KO clones at this time point (Fig. 6a,b), demonstrating that the lack of LH2 results in defective matrix mineralization.

Discussion
In this study, by generating LH2KO clones, we extensively characterized the molecular phenotypes of type I collagen. The lack of LH2 resulted in complete absence of Lys hydroxylation in all telopeptides, i.e. N-(9 N ) and C-telo (16 C ) of an α1 and N-telo (5 N ) of an α2 chains, thus, LH2 is solely responsible for hydroxylation of all Lys residues in telopeptides. Consistent with these data, the Hyl ald -derived cross-links, the major cross-links in www.nature.com/scientificreports/ MC/EV collagen, were completely absent from KO collagen and were replaced with Lys ald -derived cross-links. Moreover, our data indicated that LH2 may also be involved in helical Lys hydroxylation in a site-specific manner. The lack of LH2-catalyzed modification has significant impact on collagen solubility, collagen fibrillogenesis and matrix mineralization. In addition, LH2 could be involved in glucosylation of galactosyl Hyl. It should be noted, however, that this study was conducted using osteoblastic MC3T3 cells, thus, the phenotypes observed could be due in part to attributes of these cells.
Though the role of LH2 as telopeptidyl LH has been widely accepted 28 , the evidence reported thus far was not complete due mainly to the lack of appropriate models and analytical tools. Since LH2 KO mice die at early embryonic stage 32 , we generated LH2 KO clones using MC cells. MC cells are derived from normal mouse Table 3. Summary of site-specific modification analysis by mass spectrometry of non-cross-linked, hydroxylated and glycosylated residues in type I collagen from controls (MC and EV) and KO clones. Lys hydroxylation and its glycosylation (%) represents the relative levels of Lys, Hyl, G-Hyl, and GG-Hyl (Lys + Hyl + G-Hyl + GG-Hyl = 100%). Values represent mean ± S.D. (n = 3) of triplicate analysis for each group. Statistical differences were determined by Kruskal-Wallis one-way analysis of variance and means comparison with the controls by Dunnett's method. Lys lysine, Hyl hydroxylysine, G-galactosyl-, GG-glucosylgalactosyl, MC MC3T3-E1, EV empty vector, KO knock-out. *p < 0.05, **p < 0.01, and ***p < 0.001 between MC and KO; ## p < 0.01 and ### p < 0.001 between EV and KO, respectively.    Table 2). Values represent percentages of Lys hydroxylation calculated as Hyl/(Lys + Hyl) × 100. Hyl in the helical domain is a sum of non-glycosylated, G, and GG-Hyl (see Table 3). Lys lysine, Hyl hydroxylysine, G-galactosyl-, GGglucosylgalactosyl-, MC MC3T3-E1, EV empty vector, KO knock-out. Values represent mean ± S.D. (n = 3) of triplicate for each group. Statistical differences were determined by the method described above (Fig. 1 legend). *p < 0.05, **p < 0.01, and ***p < 0.001 between MC and KO; # p < 0.05, ## p < 0.01, and ### p < 0.001 between EV and KO, respectively. The relative levels at α1(I)K918/930 show the percentage of "Hyl + Hyl". See Table 3.  16,40 . These characteristics make MC cells an excellent model to investigate the biological functions of Lys modifications by manipulating specific LH gene expression and characterizing its effects on type I collagen 41 . Our current data unequivocally demonstrate that all Lys residues in telopeptides are hydroxylated solely by LH2, and neither LH1 nor LH3 can compensate for this function. It is not clear at this point what determines such substrate specificity for LH2. However, considering the fact that an acidic amino acid, Glu or Asp, is positioned next/close to telopeptidyl Lys residues (i.e. -Glu-Lys-Ser-in N-and C-telo of an α1 chain in both mouse and human, and -Asp-Lys-Gly-or -Asp-Gly-Lys-Gly-in N-telo of the mouse or human α2 chain, respectively), the presence of two basic Arg residues adjacent to the catalytic site of LH2 (R680 and R682) is likely important to determine such specificity 6 . Notably, these Arg residues are absent in LH1 or LH3 which explains their inability to compensate for LH2 deficiency. It is also interesting to note that, in MC/ EV type I collagen, both N-and C-telo Lys residues of an α1 chain are ~ 50% hydroxylated while the N-telo Lys      34 . In contrast, Syx et al. has stated that a mutant Hsp47, which showed a reduced binding to type I collagen, resulted in decreased LH2 44 . These inconsistent data suggest that Hsp47 may act as a positive or negative regulator of LH2 in a context-dependent manner. Interestingly, our present study showed that Fkbp65, Hsp47 and Bip protein levels were reduced in KO clones compared to MC (Fig. 2), suggesting that this chaperone complex may be destabilized by the lack of LH2.
It has been speculated that LH2 also catalyzes helical Lys hydroxylation based on its ability to hydroxylate the Lys residues in the synthetic (Ile-Lys-Gly) 3 peptide and the data from the LH2/proα1(I) co-expression in an insect cell system 22 . The results indicate that, in this system, LH2 may function as a helical LH when LH1 and 3 are absent. However, the effect of LH2 expression on Lys hydroxylation at the specific molecular loci in an α1 chain including its C-telo domain or in an α2 chain including its N-telo domain were not investigated. Recently, Gistelinck et al. has reported that, in the bone from a 4-year old patient carrying a PLOD2 heterozygous mutation, Lys in the α1(I) telopeptides was severely underhydroxylated while Lys at the helical cross-linking sites in type I collagen was normally hydroxylated 25 . Their findings are consistent with our current cell-based study showing that, when LH2 is absent, on the contrary to the changes in Lys hydroxylation in the telopetides, the extent of Lys hydroxylation in the helical domain was only minimally affected (Table 3, Fig. 3). It is important to note that, when these percentage differences are converted to the number of Hyl residues in a collagen molecule, the difference between MC/EV and KO is less than ± 0.03 residues at the cross-linking sites (α1-87, α1-918/930, α2-87, α2-933) and 0-0.2 residues at the non-cross-linking sites. Since Lys hydroxylation at the helical cross-linking sites is predominantly catalyzed by LH1 and its complex such as prolyl 3-hydroxylase 3 (P3H3), Synaptonemal Complex 65 (SC65) and CypB 9,33,45,46 , it is not surprising that absence of LH2 does not significantly affect Lys hydroxylation at these functionally critical sites in the helical domain. The significance of Lys hydroxylation at other sites in the helical domain is not well defined but, possibly, they may affect the interaction between collagen and collagen-binding proteins such as small leucine-rich proteoglycans and/or cell surface receptors such as integrins and discoidin domain receptor 2 47 .
Recently, Ishikawa and co-workers reported that the cooperation between LH1 and P3H3 is required for Lys hydroxylation in the helical domain of type I collagen, and that P3H3 may function as helical LH at specific cross-linking sites 46 . They also reported that LH2 level remained unchanged in LH1 null mice 46 . In the present study, we did not find a significant change of LH1 protein in LH2 KO clones. These findings suggest that there is no apparent direct interaction between LH1 and LH2. Thus, LH2 deficiency caused only a minute change in Lys hydroxylation in the helical domain of type I collagen.
One of the intriguing findings in the current study was that absence of LH2 affects Hyl glycosylation pattern. When the percentages of G-and GG-forms in total glycosylation forms (G-+ GG-) are calculated, KO collagen showed that at most sites, the GG-was decreased at the expense of G-form in KO type I collagen (Table 5). Recently, we have reported that LH2 potentially has galactosylhydroxylysyl glucosyltransferase (GGT) activity 6 and the current data (Tables 5, 9) supports this notion. Table 6. Summary of site-specific modification analysis by mass spectrometry of prolyl 3-hydroxylation in type I collagen from controls (MC and EV) and KO clones.     48 . The deficiency of any of these components severely affects this modification leading to severe forms of recessive osteogenesis imperfecta [49][50][51] . It has been reported that the α1 Pro-986, the major site for this modification, is hydroxylated by P3H1 49 , and another modification site, α1/2 Pro-707, mainly by P3H2 52 . In the present study, we found that the extent of P3H at these sites was slightly increased in KO clones, suggesting that LH2 may interact with the P3H complex for prolyl-3-hydroxylation at these sites (Table 6). Since LH2 interacts with CypB 33 , a P3H complex member, these slight changes could occur by the lack of this interaction.
The impact of LH2 deficiency on collagen stability, fibrillogenesis and mineralization was striking. First, collagen solubility with dilute acid and pepsin digestion were markedly increased in KO collagen, i.e. > 90% of KO collagen was solubilized by these treatments while it was only ~ 30% in control groups. The marked increases in solubility in KO collagen can be explained by the differences in the nature of the cross-links. In KO collagen, since telopeptidyl Lys is not hydroxylated, the cross-links formed are all Lys ald -derived, aldimine cross-links such as deH-HLNL and deH-HHMD. The aldimine bond is known to be labile to dilute acids, thus, readily dissociated 53 . In contrast, the Hyl ald -derived bifunctional aldimine cross-links are spontaneously rearranged to ketoamines that are stable to dilute acids. The collagens containing the stable Hyl ald -derived cross-links are also more resistant against enzymatic degradation than those with the Lys ald -derived cross-links 54,55 . Since the total number of aldehydes involved in cross-linking is only ~ 8% lower in KO collagen compared to the control, the data implies that the Hyl ald -derived cross-linking is critical to confer insolubility on type I collagen. This is likely the reason why collagen enriched in the Hyl ald -derived cross-links accumulates without being readily degraded by proteolytic enzymes in fibrosis 28,56,57 and also in desmoplastic tumors such as pancreatic ductal adenocarcinoma 58 , lung cancer 29 , breast cancer 31,59 and oral cancer 30 . Such stiffened collagen matrix may not only form a shelter for cancer cells to protect them from immune cells and anti-cancer drugs but also serve as a means for cancer cells to attach, migrate and metastasize efficiently 41,60,61 . Second, based on one experiment, fibrillogenesis in LH2 KO collagen is also affected showing smaller fibril diameters compared to those of controls. This could be due to several factors including: 1. since KO collagen is more susceptible to degradation (see above), collagen fibrils may not be able to grow, 2. altered Lys modifications (hydroxylation and glycosylation) of KO collagen may favor the association with collagen-binding proteins, such as decorin, that is known to inhibit collagen fibrillogenesis 62-64 , 3. altered post-translational modifications in KO collagen may inherently limit the growth of molecular packing into a fibril. Notably, when LH2 is overexpressed in MC cells, collagen fibrils are also smaller than controls 20 . This may indicate that the extent of LH2-mediated post-translational modifications should be kept at a certain range to establish an appropriate size of collagen fibrils in this cell culture system.
In bone, fibrillar type I collagen functions as an organizer of mineral deposition and growth [65][66][67] . Since initial mineralization appears to occur in the intermolecular channel formed by contiguous hole zones in the collagen Table 7. Levels of immature reducible cross-links (DHLNL and HLNL) and mature non-reducible cross-links (Pry and HHMD) from MC, EV, and KO clones. Total aldehydes = DHLNL + HLNL + 2 × Pyr + 2 × HHMD. Values represent mean moles/mole collagen ± S.D. (n = 3) of triplicate analysis of the hydrolysates. Statistical differences were determined by Kruskal-Wallis one-way analysis of variance and means comparison with the controls by Dunnett's method. DHLNL dihydroxylysinonorleucine, HLNL hydroxylysinonorleucine, HHMD histidinohydroxymerodesmosine, Pyr pyridinoline, MC MC3T3-E1, EV empty vector, KO knock-out. ***p < 0.001 between MC and KO; # p < 0.05, ## p < 0.01, and ### p < 0.001 between EV and KO, respectively. www.nature.com/scientificreports/ fibril 68 , the pattern of intermolecular cross-linking formed at the edge of hole zones should be critical to organize mineralization 69 . The LH2 KO collagen fibrils that contain abnormal cross-linking, highly soluble and smaller in size may not serve well as a stable template to accommodate and organize matrix mineralization. This may result in defective bone formation as seen in Bruck syndrome 1 and 2, that are caused by mutations in genes encoding LH2 chaperone FKBP65 and LH2, respectively. In addition to the structural function, LH2 may regulate cellular activities through its action on integrin β1 70 that may also impact the mineralization process. Recently, we have reported bone phenotypes of LH2 heterozygous mice (LH2 +/− ) in which LH2 expression levels are only ~ 50% of those of wild type mice (LH2 +/+ ). In this animal model, LH2 +/− femurs showed lower bone mineral density and inferior bone mechanical properties compared to those of LH2 +/+ mice 71 . When cultured, LH2 +/− osteoblastic cells mineralized poorly, which is consistent with our current study. Thus, while we cannot determine to what extent the LH2-catalyzed modification directly contributes to collagen mineralization, such modification appears to play a critical role in this process.
LH2 has two isoforms: one with an additional 63 bp-exon 13A (LH2b) and the other without (LH2a) 5 . It is generally accepted that LH2b is the telopeptidyl LH, but recently it has been reported that LH2a is also capable of catalyzing Lys hydroxylation in the telopeptides 6 . Inducing these isoforms in the LH2 KO cells separately and characterizing their collagen molecular phenotypes will provide valuable insights into their distinct and/or overlapping functions. This is now underway in our laboratory and will be the subject of the separate publication.
In conclusion, this study demonstrates that the major function of LH2 is to hydroxylate the N-(α1 and α2 chains) and C-telopeptidyl (α1 chain) Lys residues of type I collagen. The deficiency of LH2 profoundly affects collagen cross-linking, solubility, fibrillogenesis, and mineralization. These results underscore the pivotal role of the LH2-mediated post-translational modifications in the formation and function of fibrillar collagen in bone. (n = 3) from three independent experiments. Statistical differences were determined by the method described above (Fig. 1. legend) Fig. S4). Oligonucleotide pairs containing these gRNA sequences were cloned into pX335 (Addgene) that contains D10A mutant Cas9 (Cas9n) 74 , to produce pX335-mLH2-1 and -2. Evaluation of off-target effect. The specificity of the various gRNAs used in this study and their potential offtarget cleavage probabilities were initially evaluated using two different algorithms, online CRISPR RGEN Tools and Off-Spotter design prior to deploying them in MC cells. We also used the Plod2 sg RNAs as queries to search for similar sequences in the mouse genome using Cas-OFFinder (http:// www. rgeno me. net/ cas-offin der/). To test whether Plod2 sgRNAs target other genomic loci by mispairing, we performed experimental testing of five candidates selected from a search allowing up to two mismatches and two bulges. DNA primers were designed to amplify the DNA sequences containing the potential off-target sites in KO-1 using the standard PCR method (Supplementary Table S1). PCR products were separated by gel electrophoresis, and the DNA bands were isolated and extracted for Sanger sequencing. Sequencing results were aligned with mouse genome using blastn algorithm to identify potential sequence variants.

Quantitative real-time PCR.
To determine the expression of Plod2, MC, EV and KO clones were plated at a density of 2 × 10 5 cells/35 mm-dish. After 48 h, total RNA was extracted with TRIzol reagent (Invitrogen). Expression levels of Plod2 mRNA were assessed by one-step quantitative reverse transcription polymerase chain reaction (RT-PCR) with ABI Prism 7500 (Applied Biosystems). The specific probe and primers set for Plod2 was purchased from ThermoFisher Scientific (TaqMan Gene Expression Assay, Mm00478767_m1) that amplifies exon 4-5 boundary, thus, amplifying both Plod2a and Plod2b. The mRNA expression levels were normalized to beta-actin (Actb; Mm01205647_g1) and analyzed by the 2 −ΔΔCT method 75 .

Western blot analysis.
To determine the protein level, the KO clones and controls were plated onto 35-mm dishes at a density of 3 × 10 5 cells/dish. After culturing for 7 days, the cells were washed with phosphate-buffered saline (PBS), lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Sodium deoxycholate, 0.1% SDS, and 1% NP-40), centrifuged at 12,000×g and the supernatant was collected. The total protein concentration was measured by the Pierce BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's protocol. The cell lysate was mixed with Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology) was used as a secondary antibody and HRP-conjugated anti-β-actin rabbit monoclonal antibody (13E5, Cell Signaling Technology) was used as an internal control for protein loading. The reactivities of HRP were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and the chemiluminescence was scanned using an Odyssey Infrared Imaging System (LI-COR Biosciences). Quantitation of proteins was performed using the Image Studio software version 4.0 (LI-COR) with normalization to β-actin levels and was then shown as the change relative to the protein levels in MC as 1.0.
Collagen preparation for biochemical analysis. MC, KO and EV clones were cultured in α-minimum essential media (Invitrogen) containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. When the cells grew to confluence, the medium was replaced with that containing 50 μg/ml of ascorbic acid. After 2 weeks of culture, the cells/matrix layers were scraped, thoroughly washed with PBS and cold distilled water several times by repeated centrifugation at 4000×g, and lyophilized.
Collagen type analysis. Collagen was extracted and purified from lyophilized cell/matrix layer of MC, EV, and KO clones by digestion with pepsin (Sigma-Aldrich, St. Louis, MO, USA; 5 mg/ml in 0.5 M acetic acid) and salt precipitation (0.7 M NaCl in 0.5 M acetic acid) as described previously 36 . Type I and III collagens were quantified by LC-MS using SI-collagen as an internal standard 35 . In brief, SI-collagen was first mixed into the purified collagen samples, and the samples were digested with sequencing grade trypsin (Promega, Madison, WI, USA; 1:50 enzyme/substrate ratio) in 100 mM Tris-HCl/1 mM CaCl 2 (pH 7.6) at 37 °C for 16 h after heat denaturation at 60 °C for 30 min. Generated marker peptides of type I and III collagens (two peptides for each α chain; stable isotopically heavy and light ones) were monitored by LC-QqQ-MS on a 3200 QTRAP hybrid QqQ/ linear ion trap mass spectrometer (AB Sciex, Foster City, CA, USA) with an Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA) using a BIOshell A160 Peptide C18 HPLC column (5 µm particle size, L × I.D. 150 mm × 2.1 mm; Supelco, Bellefonte, PA, USA) to determine the concentrations of type I and type III collagens.

Reduction with NaB 3 H 4 .
Lyophilized cell/matrix samples (~ 2.0 mg each) were suspended in buffer containing 0.15 M N-trismethyl-2-aminoethanesulfonic acid, and 0.05 M Tris-HCl, pH 7.4, and reduced with standardized NaB 3 H 4 . The specific activity of the NaB 3 H 4 was determined by the method previously reported 76 .
The reduced samples were washed with cold distilled water several times by repeated centrifugation at 4000×g and lyophilized.

Quantification of Hyl by HPLC.
Reduced collagen was hydrolyzed with 6 N HCl and subjected to amino acid analysis 77 . The level of total Hyl in a collagen molecule was calculated based on the value of 300 residues of Hyp per collagen molecule, which were quantified as residues/collagen molecule 14 .
Site-specific characterization of post-translational modifications of type I collagen. The purified collagen samples were digested with trypsin as described above to analyze the Lys post-translational modifications at the specific molecular sites within the triple helical domain of type I collagen 37 . In addition, to analyze Lys hydroxylation at the telopeptide domains of type I collagen, the lyophilized cell/matrix samples were sequentially digested with bacterial collagenase and pepsin as previously reported 37 . In brief, the samples were digested with 0.01 mg/ml of collagenase from Grimontia hollisae (Nippi, Tokyo, Japan) 78 in 100 mM Tris-HCl/5 mM CaCl 2 (pH 7.5) at 37 °C for 16 h after heating at 60 °C for 30 min. After addition of acetic acid (final 0.5 M), the collagenase-digests were further digested with 0.01 mg/ml of pepsin (Sigma-Aldrich) at 37 °C for 16 h. The trypsin-or collagenase/pepsin-digests were subjected to LC-QTOF-MS analysis on an ultra-high resolution QTOF mass spectrometer (maXis II, Bruker Daltonics, Bremen, Germany) coupled to a Shimadzu Prominence UFLC-XR system (Shimadzu, Kyoto, Japan) using an Ascentis Express C18 HPLC column (5 µm particle size, L × I.D. 150 mm × 2.1 mm; Supelco) 37 . Site occupancy of Lys hydroxylation/glycosylation (Lys, Hyl, G-Hyl, and GG-Hyl) was calculated using the peak area ratio of extracted ion chromatograms (mass precision range = ± 0.05) of peptides containing the respective molecular species as previously reported 8,33,37,79 .